(1) Field of the Invention
This invention relates to a process and apparatus for forming solution gradients, and more particularly to a novel process and apparatus for generating a continuous solution gradient.
(2) Description of the Prior Art
Separation of macromolucules (proteins, DNA and RNA) and larger aggregates, such as viruses and cells, has been and continues to be one of the primary objectives in biochemical research. Perhaps the oldest and still most widely used separation techniques is solution gradient density centrifugation comprising three basic steps: forming a solution gradient, e.g. of sucrose in a tube; centrifuging a sample into the gradient; and recovering the now separated samples from various positions in the gradient-containing tube, sometimes referred to as fractionation.
The term "gradient" implies a continuous variation in concentration from top to bottom, e.g. 5 to 45% sucrose. The gradient performs two critical functions. First and foremost, the gradient prevents mixing in a vertical direction. During acceleration and deceleration of the tube in the centrifuge, a mild degree of mixing is induced which, if unchecked by the gradient, would thoroughly mix the contents in the tube. The gradient, however, prevents such mixing of the density differential between adjacent layers. Secondly, heavier sucrose solutions are much more viscous than light sucrose solutions and consequently, there is established a viscosity gradient. Such a viscosity gradient is useful because "g" forces are greatest at the bottom of the tube (highest radius from the center of rotation) and the increased viscosity effectively cancels the increased "g" forces giving a nearly uniform rate of molecule or particle migration from top to bottom, and consequently one can predict the position of desired molecule at the end of a run.
One of the most serious problems in the constructions of sucrose gradients is reproducibility. It is apparent that the rate of migration of any molecular species through a gradient is subject to the cumulative effects of bouyancy and viscosity of the gradient. Since these two parameters are caused by the shape of the sucrose gradient itself, tube to tube variation in the gradient will produce tube to tube variation in the final position of any molecular species. Often, it is desired to determine whether subtle changes have occurred in the size or shape of cell components, and with very reproducible gradients, such differences may be detected. By the same token, the absolute shape of the gradient is less important so long as the gradient is reprodicible.
There has been a steady but slow evolution in the techniques used to form sucrose gradients beginning with the laborious manual layering of one solution after another into a tube requiring a plurality of pipettes, a steady hand and mountains of patience and time. Such technique was quickly supplanted with a technique similar to chromatographic technology wherein two solutions, in this case the highest and lowest sucrose concentration in a desired gradient, are measured into two adjacent chambers. The mixing chamber (heavy sucrose) is connected to a centrifuge tube on one side and the other chamber (light sucrose) on the other side. As the mixing chamber's contents empty into the centrifuge tube, the contents of the other chamber enter and gradually lower their sucrose concentration. As the chambers empty, the ouflow approaches the light chamber's concentration. Such chromatography-like technology is the most commonly used technique and produces either linear or exponential gradients with minor modifications, but has two major drawbacks, i.e. time and reproducibility. When more than one gradient is desired, the outflow must be partitioned, and nothing has yet been developed that will insure exactly the same flow into each tube. Consequently, a user must watch the level in each tube, clamping off the fast ones until the slow ones catch up, etc. Additionally, there will be slight differences between the gradient in the various tubes because of constant flow adjustments.
Another technique currently in use is a freeze-thaw method wherein a homogenous solution is introduced into a centrifuge tube and the tube is subjected to a plurality of freeze-thaw cycles. Such freeze-thaw method suffers from a serious drawback in that while the freezing and thawing produces a gradient (ice floats and excludes solute molecules from the pure water matrix), any buffer is subjected to the same forces and also ends up as a gradient, producing numerous potential artificacts. Reproducibility is poor because no two tubes thaw out exactly the same way, and also because the gradients decay with time.